1. Field of the Invention
The present invention relates to a scanner type fluorescence detection apparatus for detecting fluorescence signals emitted from a specific substance in a sample and determining quantitatively the substance from the quantity of the detected signals. Particularly, the present invention relates to a scanner type fluorescence detection apparatus which is useful for real-time monitoring (monitoring of the change with time of the fluorescence signal quantity) of numerous samples in clinical diagnosis, like samples incubated at a prescribed temperature in an enzyme reaction or a like reaction.
2. Description of the Related Art
For real-time monitoring of the progress of formation of a fluorescent product in an enzymatic reaction, for example, the fluorescence from the sample is detected while the sample (liquid reaction mixture) is being incubated at a prescribed temperature. In clinical diagnosis, the detection should be conducted rapidly for a large number of samples simultaneously.
In a first method employed conventionally in clinical diagnosis, the samples are conveyed along a temperature-controlled guide and the fluorescence is detected successively. For example, a guide is made of a highly heat-conductive material like an aluminum alloy; the temperature of the guide is controlled by a heater or a like means; the samples are conveyed one by one or in plural at a time successively by a chain, a turn table, or the like along the guide; and the fluorescence signal is detected successively by a fluorescence detector arranged along the guide.
In a second method, for example, a connected sample vessel or a titer plate which is capable of holding numerous samples is placed on a temperature-controlling means, and the fluorescence of the numerous samples is detected simultaneously. Such a system is characterized by (1) plural photosensors, (2) a multi-channel type photosensor, or (3) a mechanical moving means for moving a photosensor or a light guide (a means for introducing the fluorescence signals emitted from the sample vessels) such as optical fibers.
The apparatus employing the plural photosensors (1) requires photosensors in number corresponding to the number of the samples to be detected simultaneously, and the fluorescence signals are detected separately for the respective samples. In such a system generally, the excitation light is split and the split rays are introduced through light guides to the respective samples.
The aforementioned multi-channel type photosensor (2) employs an image sensor such as a CCD and a photodiode array instead of the plural photosensors. The image sensor detects the fluorescence signals emitted by the arrayed samples are detected as an image with retention of the light-emitting positional relations. In such a system also, the excitation light is generally split and the split rays are introduced through light guides (optical instrument or optical fibers) to the respective samples.
The aforementioned mechanical moving means (3) moves a photosensor mechanically over the plural samples, or moves the respective samples successively to the fluorescence detection position where the fluorescence is detected by the photosensor. In such a system frequently, a light guide is moved mechanically. In this constitution, a light guide for the excitation light and another light guide for the fluorescence are employed and the sample sides of the both guides are combined and are moved together to excite the samples and detect the fluorescence therefrom successively.
For solving the problems mentioned later which are involved in the above systems, another scanner type fluorescence detection apparatus is disclosed in Japanese Patent Application No. 10-254913. In this apparatus, as shown in FIG. 3, sample vessels are deployed and arranged along a circle line, and a ring portion of a ring-shaped light guide is opposed close thereto with interposition of a partition plate. An optical means for excitation light and an optical means for fluorescence light are fixed to the partition plate, and are rotated together with the partition plate. Fluorescence signals collected from the respective samples are transmitted through the ring-shaped light guide to the photosensor.
Conventional fluorescence detection apparatuses have the problems mentioned below in the real-time monitoring of the change with time of fluorescence emitted from a specified substance contained in a sample incubated at a prescribed temperature.
The aforementioned first method, in which the samples are conveyed along a temperature-controlled guide and the fluorescence is successively detected, may cause insufficient temperature-control accuracy, limitation of the speed of the treatment of a number of samples, and carry-over (contamination of the samples by sample splashing), disadvantageously. In other words, it is difficult to keep the entire of the sample delivery guide at a uniform temperature and to make uniform the thermal conduction between the delivery guide and the samples throughout the guide. Consequently, the temperature of the samples may vary during the delivery, or may differ between the samples. Further, in this method, in monitoring the change of the fluorescence signals for a long time, the same samples are delivered repeatedly, the fluorescence of the delivered samples is detected one by one successively, thereby the number of the treated samples being limited. Moreover, the carry-over cannot be prevented completely.
The aforementioned second method may cause different problems although the problems caused in the first method are solved.
The method employing the plural photosensors (1) requires high cost owing to the plural photosensors, and an installation space corresponding thereto. For size reduction of the apparatus, the number of the photosensors should be reduced for the limited installation space, which limits the number of the sample treated at one time. A photosensor of a small size such as a photodiode has not sufficient sensitivity to the faint fluorescence. The plural photodiodes should be calibrated individually. Further, since the intensity of the fluorescence signal is proportional to the intensity of the excitation light, the splitting of the excitation light from the light source will lower the detection sensitivity.
The method employing the multi-channel type photosensor (2) is not suitable because of low sensitivity to faint fluorescent light. To raise the sensitivity, an element (so-called image intensifier or the like) can be employed which amplifies the light quantity through electronic amplification by a micro-channel plate. However, this is extremely costly and is used only in special researches. Furthermore, this system detects the fluorescence over a broad range as an image, which may give rise to disadvantages of nonuniformity of light quantity detection caused by lens aberration and the additional treatment of an enormous amount of data.
The method employing the mechanical movement means (3) is restricted in movement range of the light guide by the limitation by flexibility of the light guide and may cause disconnection. In this method, the light transmission efficiency of the light guide is varied by flection, which makes difficult the detection of fluorescence with high reproducibility. The mechanical movement of the photosensor is also limited in the movement range by the attached cables, and the cable may cause disconnection.
The scanner type fluorescence detection apparatus disclosed in aforementioned Japanese Patent Application No. 10-254913, which solves the above problems, becomes larger in size of the apparatus and higher in cost with the increase of the number of the sample vessels to be held, disadvantageously. Specifically, the ring-shaped input end is counterposed close to the sample vessels arranged along a circle line with interposition of a partition plate. Therefore, the diameter of the ring constituted of optical fibers should be made larger with the increase of the number of the held sample vessels, requiring a larger number of the employed optical fibers. Further, the fluorescence signal output end of the light guide is also made thicker, which enlarges the areas of fluorescence wavelength selection means such as the light sensing face of the photosensor and an interference filter. The larger sizes and higher cost of the light guide, the photosensor, interference filter, and other optical means result in significant larger size and higher cost as a whole.
As described above, the fluorescence detection apparatus for real-time monitoring of fluorescence signals, especially for real-time monitoring of samples incubated at a prescribed temperature, should satisfy the requirements of (A) precise temperature control, (B) quick treatment of many samples, (C) high sensitivity, (D) high reliability (less mechanical trouble such as disconnection and failure of movable parts, higher reproducibility in fluorescence detection, low possibility of carry-over), (E) lower cost (simpler constitution of the apparatus, use of less expensive parts for data treatment, etc.), and (F) a smaller size of the apparatus.
The present invention intends to provide a fluorescence detection apparatus satisfying the aforementioned requirements. Specifically the present invention intends to provide a fluorescence detection apparatus for real-time monitoring of stationarily held numerous samples without increase of the size and cost of the apparatus for a larger number of the treated samples. The present invention intends further to provide the fluorescence detection apparatus having additionally an incubation function.
The fluorescence detection apparatus of the present invention comprises a sample holder for holding stationarily sample vessels deployed along a circle line or concentric circle lines having different radiuses, a partition plate connected to a driving means to be rotatable around the center of the circle line or concentric circle lines, an optical means for excitation light and an optical means for fluorescence light fixed respectively to the partition plate to be rotatable in integration therewith, a first light guide constituted of numerous optical fibers, a photosensor, and a light source for generating the excitation light, wherein
(a) the optical means for excitation light is placed to introduce the excitation light from the side of the rotation center of the partition plate to excite selectively a sample in one of the sample vessels,
(b) the optical means of fluorescence are provided in number of the circle lines having different diameters for arrangement of the sample vessels, and have respectively at least one second light guide to collect the fluorescence signals from the samples on the respective circle lines,
(c) the optical fibers of the first light guide are deployed to confront the circular locus or loci drawn by fluorescence signal output ends of all of the optical means for fluorescence light on rotation of the shield plate, and are arranged densely to confront the photosensor at the fluorescence signal output end, and
(d) the excitation light is successively introduced, with rotation of the partition plate, to the respective sample vessels arranged along the circle lines, and simultaneously the fluorescence is detected through the optical means for fluorescence light including the second light guide.
Another embodiment of the fluorescence detection apparatus has a single optical fiber or plural optical fibers as the second light guide.
Still another embodiment of the fluorescence detection apparatus of the present invention has the optical fiber constituting the first light guide deployed and arranged continuously in a ring form at the fluorescence signal input end, having the ring center on the shield plate.
A further embodiment of the present invention comprises an insulation vessel for housing at least the sample holder, and a temperature control means for keeping the samples at a prescribed temperature.